Nanoparticles of cerium oxide targeted to an amyloid-beta antigen of Alzheimer&#39;s disease and associated methods

ABSTRACT

Disclosed is a composition immunologically targeted to Alzheimer&#39;s disease (AD), the composition containing amine functionalized nanoparticles of Cerium oxide coated with polyethylene glycol and bearing an antibody specific for an amyloid-beta antigen associated with AD. The invention also includes a medication manufactured with the targeted nanoceria particles and methods of treatment by administering the targeted nanoceria particles to patients in need thereof.

RELATED APPLICATION

This application claims priority from copending provisional applicationSer. No. 61/383,773, which was filed on 17 Sep. 2010, and which isincorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

The invention claimed herein was made with at least partial support fromthe U.S. Government. Accordingly, the government may have certain rightsin the invention, as specified by law.

FIELD OF THE INVENTION

The present invention relates to the field of neurological diseases and,more particularly, to a cerium oxide nanoparticulate composition usefulin treatment of Alzheimer's disease.

BACKGROUND OF THE INVENTION

Current therapies for Alzheimer disease (AD) provide moderatesymptomatic delay at various stages of the disease, but do not arrestthe disease progression, and hence, new approaches to the diseasemanagement are urgently needed.

In recent years, cerium oxide nanoparticles have been studied aspossible potent antioxidant agents that might be able to exertneuroprotective effects. We herein disclose the specific design of atargeted nanoceria-based formulation suitable for AD therapy. The testresults obtained indicate the present composition is useful forselective delivery of immunonanoparticles to Aβ (amyloid-beta) plaqueswith concomitant rescue of neuronal survival and neurite dystrophy. Theformulation appears to work by regulating the expression of the BDNFsignal transduction pathway.

Oxidative stress and amyloid-beta (Aβ) are considered major etiologicaland pathological factors initiating and promoting neurodegeneration inAlzheimer's disease (AD) due to the production of free radicals (1-6).To date, use of multiple doses of antioxidants has met with onlyllimited success in abolishing these pathological conditions (7).

Recently, we have discovered that cerium oxide nanoparticles (CNPs) areredox active and biocompatible with both superoxide dismutase (8) andcatalase mimetic activity (9). Among the lanthanide series of elements,cerium is distinctive in that it has two partially filled subshells ofelectrons, 4f and 5d, with many excited substates, resulting in avalence structure that undergoes significant alterations depending onthe chemical environment (10-13). A predominant +3 oxidation state onthe surface of CNPs is responsible for the nanoparticles' uniqueantioxidant properties (14, 15). We have shown that a single dose ofCNPs prevents retinal degeneration induced by peroxides (16). In vitro,one low dose maintained radical scavenging and protective effects forlong durations and multiple insults, suggesting the possibility of itsregenerative activity. Therefore, CNPs have been investigated aspossible nanopharmacological composition for use against diseasesassociated with oxidative stress (17-22).

Previously, on an AD human in vitro model, we have confirmed theanti-oxidant properties of CNPs. We have also demonstrated that CNPs donot act as mere anti-oxidant agents, but that they seem regulate signaltransduction pathways involved in neuroprotection (23) To date, no earlybiomarkers for AD have been identified, therefore the appearance ofsymptoms is indicative of the full-blown disease.

The present disclosure concerns a novel approach wherein a CNPformulation comprises specifically targeted nanoparticles able to directonly to such targets as the brain areas of neurodegeneration and toexert specific effects counteracting neurite dystrophy and inhibitingdisease progression. The presently disclosed composition is active atsignificantly lower dosages and in a single administration as opposed tofree nanoparticles. This improvement was achieved by the synthesis ofCNPs through the development of an improved method for the conjugationof anti-amyloid β antibodies to the nanoparticles with selectivedelivery to Aβ plaques and a concomitant increase of neuronal survival.

SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention advantageouslyprovides a composition of polyethylene glycol (PEG) coated nanoparticlesof Cerium oxide having an antibody bound thereto, the antibody beingspecific for an antigen associated with a predetermined diseasecondition. Preferably, the nanoparticles are amine functionalized priorto coating, so as to promote coating by the PEG. In a preferredembodiment of the invention, the antibody is specifically targetedagainst an amyloid-beta antigen associated with a neurodegenerativedisease. The nanoparticles are approximately from 3-5 nm in size priorto coating with PEG. The composition may be contained in a manufacturedmedication biologically acceptable for administration to a patientexhibiting symptoms of the predetermined disease. Accordingly, a methodof treatment for the predetermined disease condition includesadministration of the composition to a patient in need thereof.

More specifically, the present invention provides for a compositionspecifically targeted to a neurodegenerative disease, said compositioncomprising amine functionalized nanoparticles of Cerium oxide coatedwith polyethylene glycol and bearing an antibody specific for an antigenassociated with the neurodegenerative disease. As noted above, thiscomposition may also be contained in a manufactured medicationbiologically acceptable for administration to a patient exhibitingsymptoms of the neurodegenerative disease. Included in this preferredembodiment of the invention is a method of treatment for aneurodegenerative disease, the method comprising administering thisvariation of the composition to a patient in need thereof.

A more specific yet embodiment of the present invention includes acomposition immunologically targeted to Alzheimer's disease (AD), thecomposition comprising amine functionalized nanoparticles of Ceriumoxide coated with polyethylene glycol and bearing an antibody specificfor an amyloid-beta antigen associated with AD. This composition may becontained in a manufactured medication biologically acceptable foradministration to a patient suffering from AD. This particularembodiment also includes a method of treatment for Alzheimer's disease,the method comprising administering the composition to a patientsuffering from AD.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings,presented for solely for exemplary purposes and not with intent to limitthe invention thereto, and in which:

FIG. 1 presents the characterization of nanoceria: (A) HRTEM image ofmicroemulsion nanoparticles showing control particle size distribution(3-5 nm) consistent to our group's well established synthesis procedure(42). The large d spaced planes (111) primarily easily focused at 300 kVis indicated in micrographs. Smaller d spacing planes (220,311) are notmarked for clear representation of micrographs. The inset shows theselected area electron diffraction pattern of nanoparticle captured atlow magnification confirming the crystalline nature and fluoritestructure of CNPs by calculation of each diffraction ring diameter(Lλ=RD). (B) The amine functionalization of CNPs was confirmed by XPS.In the XPS two O (1s) peak corresponding two different valance state ofceria (Ce³⁺ corresponds to 531.5 eV and Ce⁴⁺ corresponds to 530.6 eV)and other two confirms the functionalization. Peak 1 is O—C bond thatconnects epichlorohydrin to the cerium oxide (534.00 eV) and peak 2 isepichlorohydrin's epoxy group (533.35 eV). (C) Shows change in theappearance of CNPs (yellow) after amine functionalization (light yellow)and PEG-conjugation (dark brown); (D) UV-VIS absorbance of ceria,NH₂-functionalized ceria and PEG coated CNPs, showing the shift in theabsorbance maximum to red and blue shift after amine functionalization(38.45 nm) and PEG-conjugation (33.41 nm) (E) FTIR spectra of PEGconjugated cerium oxide nanoparticle confirming presence of PEG on CNPs;(F) SOD mimetic activity of PEG-CNPs as compared to control, showingthat surface PEG-CNPs is still active and scavenges the radicalefficiently.

FIG. 2: SMFS measurements of Aβ-protein with bare CNP in aqueous medium;deflection-extension spectrum (A) and corresponding force-extension ofAβ with silicon substrate (B), interaction force of Aβ with bare ceriananoparticle (C), amine functionalized CNPs (D), PEG-conjugated CNPs(E), Force histogram of Aβ with bare CNPs (F) and amine functionalizedGNPs (G). Multiple SMFS were conducted for force and histogram on eachsample. The total number of force and length values analyzed were n=204(for F) and n=320 (for G).

FIG. 3 Plaque presence is evaluated by ThT staining in control (B), Aβtreated (E) cells and Aβ-CNP-Ab (H). The ability of the conjugate torecognize the plaque is evaluated by TRITC labeled anti rabbit secondaryantibody in control (C), Aβ-treated (F) cells and Aβ-CNP-Ab-treatedcells (I). The ability of CNP-Ab to target only the plaque is shown inL, that is the merge of H and I. The nuclei are stained with DAPI incontrol (A), Aβ treated (D) cells and Aβ-CNP-Ab-treated cells (G).Bar=17 nm.

FIG. 4 Panel A shows cell viability evaluated by MTS assay, in control(CTR), Aβ treated (Aβ) cells and Aβ-CNP-Ab-treated cells. Data aremeans±SE, N=6, p=0.025, *p≦0.05; *, expressed vs control value. Panel Bshows apoptotic cell death, evaluated as nucleosome concentration incontrol (CTR), Aβ treated (Aβ) cells and Aβ-CNP-Ab-treated cells. Dataare means±SE, N=3, p=0.001, **p≦0.005*, expressed vs control value.Panel C shows cell death, evaluated by DAPI nuclear staining and bycounting condensed nuclei in control, Aβ treated cells andAβ-CNP-Ab-treated cells Data are means±SE, N=3, p=0.03, *p≦0.05; *,expressed vs control value. Bar=17 um. Panel D: Phase-contrastmicroscopy in control (CTR), Aβ treated (Aβ) cells and Aβ-CNP-Ab-treatedcells. Bar 17 nm; On the left the neurite number in control, Aβ treatedcells and Aβ-CNP-Ab treated cells is reported. Histograms reportn°-neurites/n°-cells. Data are means±SE, N=3, p=0.024, *p≦0.05. On theright the neuritic length in control, Aβ treated cells andAβ-CNP-Ab-treated cells. The neurite length was determined as neuritelength/Ø soma. Data are means±SE, N=4, p=0.000, ***p≦0.0005. *,expressed vs control value.

FIG. 5 Subcellular localization of the neuronal differentiation markersβ-tubulin III (β-TubIII) (green) and PPARβ/õ (red) in control (A, B; C),Aβ-treated (D, E, F) cells and Aβ-CNP-Ab (G, H, I) treated cells. In C,F and I the merged images are shown. Bar=17 nm. In the bottom westernblotting and densitometric analyses for PPARβ/õ in control (CTR),Aβ-treated (Aβ) cells and Aβ-CNP-Ab-treated cells. Band relativedensities were determined against most evident band of PVDF membraneComassie Blu stained. Data are means±SE, N=3, p=0.009, *p≦0.05. *,calculated vs control value.

FIG. 6 The upper panels show immunofluorescence analysis for NF-H 200 incontrol (A, D), Aβ-treated (B, E) cells and Aβ-CNP-Ab (C, F) treatedcells. D, E, and F are higher magnification pictures. Bar=17 μm. Nucleiwere stained with DAPI. Panels below show immunofluorescence analysisfor GAP43 in control (G, L), Aβ-treated (H, M) cells and Aβ-CNP-Ab (I,N) treated cells. L, M, and N are higher magnification pictures. Bar=17μm. Nuclei were stained with DAPI.

FIG. 7 Panel A shows western blotting and densitometric analyses forTrkB, P75NTR and cytoplasmatic pro-BDNF, in control (CTR), Aβ-treated(Aβ) cells and Aβ-CNP-Ab-treated cells Band relative densities, weredetermined against most evident band of PVDF membrane Comassie Blustained. Data are means±SE, N=3, for TrkB p=0.04, *p≦0.05. For P75NTRp=0.004, **p≦0.005; For pro-BDNF p=0.005, **p≦0.005 The * is calculatedvs control (CTR) value. Panel B shows western blotting and densitometricanalyses for extracellular and soluble pro-BDNF form assayed byimmunoprecipitating the culture media from control, Aβ-treated andAβ-CNP-Ab-treated cells. The immunoprecipitation assay was performedallowing the collected media to be absorbed on Protein A Sepharose ACL-4B followed by a precipitation step with a specific anti-BDNFantibody Band relative densities were determined using TotalLab software(ABEL Science-Ware srl, Italy) and values were given as % over control.Data are means±SE, N=3, p=0.009, *p≦0.05* is calculated vs controlvalue. Panel C shows western blotting and densitometric analyses forp-ERK1,2 and p-ERK5, in control, Aβ-treated cells and Aβ-CNP-Ab-treatedcells. Band relative densities, were determined against most evidentband of PVDF membrane Comassie Blu stained. Data are means±SE, N=3, forp-ERK1: Aβ vs CTR p=0.004, **p≦0.005; Aβ-CNP-Ab vs CTR p=0.001,**p≦0.005. For p-ERK2: Aβ vs CTR p=0.001, **p≦0.005. Aβ-CNP-Ab vs CTRp=0.016, *p≦0.05. For p-ERK5: Aβ vs CTR p=0.012, *p≦0.05; Aβ-CNP-Ab vsCTR p=0.000, ***p≦50.0005. * is calculated vs control value.

FIG. 8 shows the diffraction pattern of nanoceria showing typical peakbroadening indicative of nanocrystalline particles.

FIG. 9 depicts the results of Energy dispersive X-ray spectroscopyillustrating the elemental composition which confirms that CNPs areessentially free of impurities.

FIG. 10 portrays the conjugation of Anti-β-amyloid antibody with NH2terminal PEG CNP.

FIG. 11 shows the ligation of the specific antibody against Aβ to thenanoparticles allows the CNPs-Ab to specifically recognize the plaque,having minimum or no interaction with nearby neuronal cells. This notionis supported by the effects observed when the same antibody, utilizedfor the ligation, is utilized alone to treat the cells after the Aβchallenge; in fact, it identifies the plaque (A, B, C) but havingminimum or no effect of cell viability (D).

FIG. 12 presents preliminary experimental results showing the effects oncell viability of PEG-CNP or NH2-CNP administered alone or after A-betachallenge indicated that PEG-CNP are more effective than NH2-CNP inpromoting neuronal survival. Nevertheless CNPs-PEG-Ab were found to bemore effective as compared to PEG-CNP.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown.

Unless otherwise defined, all technical and scientific terms used hereinare intended to have the same meaning as commonly understood in the artto which this invention pertains and at the time of its filing. Althoughvarious methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. However, the skilledshould understand that the methods and materials used and described areexamples and may not the only ones suitable for use in the invention.

Moreover, it should also be understood that any temperature, weight,volume, time interval, pH, salinity, molarity or molality, range,concentration and any other measurements, quantities or numericalexpressions given herein are intended to be approximate and not exact orcritical figures unless expressly stated to the contrary. Accordingly,where appropriate to the invention and as understood by those of skillin the art, it is proper to describe the various aspects of theinvention using approximate or relative terms and terms of degreecommonly employed in patent applications, such as: so dimensioned,about, approximately, substantially, essentially, consisting essentiallyof, comprising, and effective amount.

Further, any publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety as if they were part of this specification. However, in case ofconflict, the present specification, including any definitions, willcontrol. In addition, the materials, methods and examples given areillustrative in nature only and not intended to be limiting.

Accordingly, this invention may be embodied in many different forms andshould not be construed as limited to the illustrated embodiments setforth herein. Rather, these illustrated embodiments are provided so thatthis disclosure will be thorough, complete, and will fully convey thescope of the invention to those skilled in the art. Other features andadvantages of the invention will be apparent from the following detaileddescription, and from the claims.

Methods

Synthesis and characterization of CNPs CNPs of approximate size of 3-5nm were synthesized by a microemulsion method describe elsewhere (36).After preparation, the particles were washed with acetone and water forsix to eight times to remove the surfactant and other impurities. Highresolution transmission electron microscopy (HRTEM), with FEI Tecnai F30having an energy dispersive X-ray (EDX) analyzer, was carried out tostudy the size and morphology of the nanoparticles.Amine Functionalization of CNPs

Prepared CNPs were suspended in 0.1 M NaOH solution and stirred for 5minutes. Five milliliters of distilled epichlorohydrin and 0.5 mL of 2 MNaOH were added and stirred at room temperature. The nanoparticles werethen recovered by centrifugation and washed with water several times.Next, the nanoparticles were suspended in water and 30% ammoniumhydroxide solution was added and stirred for several hours. Finally, theresulting amine functionalized nanoparticles were recovered bycentrifugation, washed with water (three to four times), and dried (37).

A 5400 PHI ESCA (XPS) spectrometer was used to obtain XPS data toconfirm the amine functionalization. The base pressure during XPSanalysis was 10-10 Torr and Mg—Kα X-radiation (1253.6 eV) at a power of350 watts was used.

Preparation of PEG-CNPs

Polyethylene glycol (PEG) spacers with carboxy and amine terminals andhaving a spacer arm of 18.1 Å were selected for the study. We chosebi-functional PEG, so that one end can connect to an aminefunctionalized nanoparticle and the other end to the antibody. Thecarboxy terminal of the bi-functionalized PEG molecule was coupled tothe amine functionalized CNPs using EDC and Sulfo NHS couplingchemistry. 1 mg/ml CA(PEG)₄ was dissolved in 0.05M NaCl, pH 6 buffer. 2mM EDC and 5 mM Sulfo-NHS were added to the CA(PEG)₄ solution andstirred at room temperature. Amine functionalized CNPs were resuspendedin sodium phosphate buffer, added to the reaction mixture and stirred.The molar ratio of amine functionalized ceria:CA(PEG)₄ was 1:4, used forthe reaction. PEG-CNPs were recovered by centrifugation, washed withwater (three to four times), and dried. UV-Visible spectroscopy andFourier transform infrared (FTIR) spectra were obtained to confirm thePEG molecule on the nanoparticle surface using PerkinElmer Lamda750S andPerkinElmer Spectrum, respectively. Superoxide dismutase (SOD) mimeticactivity of the PEG-conjugated CNP was estimated using SOD Assay kit(Sigma-Aldrich Corp., St. Louis, Mo., USA) according to the manufacturerinstructions.

Conjugation of Aβ Antibody with PEG-CNP

In the first step, sodium azide and other salt were removed from theanti Aβ antibody by centrifuging through 10 kD cut-off Centricon®(Millipore Inc.). The antibody (1 mg/ml concentration) was diluted inNaCl, pH 6 buffer. 2 mM EDC and 5 mM Sulfo-NHS were added to theantibody solution and stirred at room temperature. PEG-CNPs wereresuspended in sodium phosphate buffer, added to the reaction mixtureand stirred. The molar ratio of amine functionalized antibody toPEG-CNPs used for the reaction was about 1:5. PEG-ceria nanoparticleswere recovered by centrifugation, washed with water (three to fourtimes), and re-suspended in distilled water. The concentration of ceriaafter antibody conjugation was assayed by UV-Visible spectroscopy.Bradford assays were performed to confirm antibody conjugation to thePEG-ceria nanoparticle.

Cell Cultures

SH-SY5Y cells (ATCC) were seeded at about 1×10⁴ cells/cm² and culturedfor 7 divisions (DIV) in FBS-free RPMI 1640 differentiating mediumcontaining N2 supplement in order to promote neuronal differentiation.

Aβ Fibril Formation

Aβ(25-35) is frequently used in investigating Aβ properties as a lessexpensive and more easily handled substitute for the native full-lengthpeptide, Aβ(1-42). Indeed, Aβ(25-35) mimics the toxicological andaggregation properties of the full-length peptide, though thesecharacteristics are enhanced; i.e., the shorter peptide is more toxic tocultured neurons, exhibits earlier toxicity, causes more severe membraneprotein oxidation, and aggregates faster than the native Aβ(1-42) (38).

The amyloid fibrils were obtained as previously described (39).Specifically, the Aβ(25-35) stock solution (500 μM) was prepareddissolving Aβ in FBS-free differentiating medium containing N2supplement (pH 7.4) and stored at −20 C.°. The amyloid fibrils wereobtained incubating Aβ(25-35) stock solution at 37° C. for 8 days.

Fluorimetric Assay

The amyloid polymerization status was checked by the thioflavin T (ThT)fluorescence method before each treatment (40). ThT binds specificallyto amyloid fibrils, and such binding produces a shift in its emissionspectrum and an increase in the fluorescent signal, which isproportional to the amount of amyloid formed (41-43). Followingincubation, Aβ in 20 mM Tris HCl Buffer, pH 8.0, and 1.5 μM ThT in afinal volume of 2 ml were analyzed. Fluorescence was monitored byspectrofluorimetry at an excitation wavelength of 450 nm and an emissionwavelength of 485 nm, as previously described (41).

Treatments

Differentiated cells were treated with Aβ25-35 (12.5 μM, f.c.) for 24 h.For nanoparticles treatment, cells were subjected for 4 h to acutechallenge with Aβ25-35, and were treated with an effective dose (200 nM,f.c.) of cerium oxide nanoparticles conjugated to anti-Aβ antibody(CNPs-Ab).

Aβ Plaque Detection

In order to asses if the ligation of antibody to the nanoparticlesallows them to bind specifically to Aβ plaques, double immonoflorescencestaining was performed. Briefly, cells, grown on coverslips, were fixedin 4% paraformaldehyde in PBS for 10 min at RT. Cells were thenincubated with 0.05% solution of ThT and Tritc-labeled antirabbit IgGsecondary antibody (1:100), for 20 min at RT. Nuclei were counterstainedwith DAPI (300 ng/ml). After extensive washings, coverslips were mountedwith Vectashield mounting medium and photographed in a fluorescencemicroscope (AXIOPHOT, Zeiss).

Cell Viability and Death

Cells, plated in 24 multiwell plates, were incubated after treatmentsfor 2 h with CellTiter 96 AQueous One Solution, a imetric viability testmethod based on3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenil)-2-(4-sulfophenyl)-2H-tetrazolium(MTS). The quantity of formazan formed, as a function of viability, wasmeasured at 490 nm using an ELISA plate reader. All MTS assays wereperformed in triplicate.

After Aβ exposure, cells on coverslips were fixed in 4% paraformaldehydeat room temperature for 10 min, then stained with DAPI (300 ng/ml) for20 min and examined under UV illumination using a fluorescencemicroscope. To quantify the apoptotic process, nuclei with bothfragmented or condensed DNA and normal DNA were counted. Fivefields/coverslips were counted. Data, from 3 different experiments, areexpressed as a percentage of the total cells counted. For apoptosisdetection, cells were seeded in 24-well plates at a density of 1×10⁴cells/cm². Control and treated cells were analyzed for apoptosis usingthe cell death detection ELISA kit for the nucleosome detection.Absorbances at 405 nm with respect to 490 nm were recorded according tomanufacturer's directions.

Morphometry

Control and treated cells, grown on coverslips, were fixed in 4%paraformaldehyde in PBS for 20 min at RT. After washings, coverslipswere mounted with Vectashield® and phase-contrast observations wereperformed by an AXIOPHOT Zeiss microscope, equipped with a micrometricocular lens. The processes longer than the cell body mean diameter (Ø),which should be regarded as neurites, were counted and the results wereexpressed as neurite number vs. the total cell number. The neuritelength was determined by comparing the neurite length with the meandiameter (Ø) of cell soma and reported as neurite length/soma (Ø).

Immunofluorescence

Control and treated cells, grown on coverslips, were fixed in 4%paraformaldehyde in PBS for 20 min at RT and permeabilized in PBScontaining 0.1% Triton X-100 for 5 min at RT. Cells were then incubatedwith mouse anti-β-tubulin III (1:300) and anti PPARβ (1:100) diluted inPBS containing 3% BSA overnight at 4° C. The immunolocalization ofGAP-43 and heavy neurofilament (NF-H) was performed by permeabilizingthe fixed cells with absolute methanol for 10 min at −20° C. After that,cells were rehydrated with PBS for 5 min and incubated with anti-GAP 43(1:300) and anti-NF-H, (1:200) antibodies, overnight at 4° C. Afterextensive washings with PBS, cells were treated with fluorescein-labeledanti-mouse or Tritc-labeled anti-rabbit IgG secondary antibodies (1:100in PBS containing 3% BSA) for 30 min at RT. Nuclei were counterstainedwith DAPI (300 ng/ml). After extensive washings, coverslips were mountedwith Vectashield mounting medium and photographed in a fluorescencemicroscope (AXIOPHOT, Zeiss).

Western Blot

Cells were washed in ice-cold PBS and homogenized in ice-cold RIPAbuffer (10 mM Hepes, pH7.4, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mMdithiothreitol) with a protease inhibitor mixture (100 mg/mlphenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, 2 mM leupeptin, and 1mg/ml pepstatin). The lysate was subjected to centrifugation at 600×gfor 30 min at 4° C., and the supernatant was collected. Samples (25-50μg/lane) were analyzed by 10% SDS-PAGE, transferred to PVDF membranes,and blocked in Tris-buffered saline containing 5% non fat milk, and 0.1%Tween20®. Membranes were incubated with different primary antibodies,anti-PPARβ (1:1000), anti-BDNF (1:200), anti TrkB (1:200) anti p-75 NTR(1:100), anti p-ERK1,2 (1:200), anti-ERK1,2 (1:1000), anti-p-ERK5(1:200) overnight at 4° C. and then probed with horseradishperoxidase-conjugated mouse or rabbit secondary antibodies (1:1000).Immunoreactive bands were visualized by chemiluminescence. Band relativedensities, against most evident band of PVDF membrane Comassie Blustained, were determined using TotalLab® software (ABEL Science-Waresrl, Italy) and values were given as relative units.

Immunoprecipitation Assay

The immunoprecipitation assay was performed to estimate the amount ofbrain-derived neurotrophic factor (BDNF) protein in the culture mediaobtained from control and treated cells. To immunoprecipitate BDNF, asolution of 100 μg Protein A Sepharose A CL-4B was added to an equalamount (1000 μg of total protein) of supernatant samples collected fromeach condition. The suspension obtained was allowed to shake for 2 h at4° C. and incubated with 5 μg of anti-BDNF primary antibody overnight at4° C. The immunoprecipitates were then collected by centrifugation andthe supernatant aspirated and discarded. Resuspended pellets weresubjected to 15% SDS-PAGE, transferred to PVDF membranes and probed withspecific anti BDNF antibody. After incubation with horseradishperoxidase-conjugated rabbit secondary antibodies (1:1000), theimmunoreactive bands were visualized by chemiluminescence. Band relativedensities were determined using TotalLab® software (ABEL Science-Waresrl, Italy) and values were given as percentage over control.

Statistics

Experiments were performed at least in triplicate. Data were representedas means±Standard Errors. Where appropriate, one-way ANOVA test followedby Scheffe's post hoc test analysis was performed using SPSS software. Pvalues less than 0.05 were considered statistically significant.

Results

Functionalization and Characterization of CNPs

FIG. 1 shows the characterization of synthesized CNPs. In FIG. 1(A)depicts a TEM image showing well dispersed nanoparticle with a sizedistribution ranging approximately between 3-5 nm, (inset: X-raydiffraction pattern). The diffraction pattern showed typical peakbroadening indicative of nanocrystalline particles (Suppl materials,Figure S1). X-ray spectroscopy illustrates the elemental compositionwhich confirms that CNPs are free from any impurities (Suppl materials,Figure S2). FIG. 1B represents the high resolution XPS spectra of aminefunctionalized CNP, showing the evidence of complex formation. Peak 1shows an O—C bond and represents the epichlorohydrin molecule attachmentto the nanoparticle (534.00 eV) and peak 2 corresponds toepichlorohydrin's epoxy group (533.35 eV). The other two peaks are dueto the presence of mixed valence state of cerium atom (Ce+3: 531.5 eV,Ce+4: 530.6 eV). FIG. 1C shows the change in the color of CNPs (darkyellow) after amine functionalization (light yellow) and PEG-conjugation(brown). Panel D presents the UV-Visible spectroscopy data showing ashift in the absorption spectrum of CNP following functionalization withNH2 (38.45 nm) and PEG (33.41 nm). The FTIR spectra (Panel E) ofPEG-CNP, confirming the PEG conjugation; SOD mimetic activity of PEGcoated CNP is presented in Panel F.

The conjugation of Anti-β-amyloid antibody with NH2 terminal PEG CNP isillustrated in scheme (Suppl materials Fig S3). The anti Aβ antibody wasattached to the PEG-CNP using EDC/Sulfo NHS coupling reaction; the tableshows the amount of antibody (μg/ml) in 5 mM PEG-CNP and CNP-NH2estimated by Bradford assay.

Single Molecular Force Spectroscopy of CNPs

We selected Aβ1-42 as a model protein to find out the interaction of theprotein with bare and functionalized CNPs. FIG. 2 shows the singlemolecular force microscopy (SMFS) measurements carried out using aSuccinimmide functionalized Silicone nitrite AFM tip (spring constant0.01 N/m and tip radious of curvature 10 nm) coated with AF protein withbare CNPs. The figure shows the deflection-extension spectrum (A) andthe corresponding force-extension (B) of Aβ with silicon. FIGS. (C), (D)and (E) show the interaction of Aβ with bare, NH2 and PEG functionalizedCNPs, respectively. Attraction force of interaction was found in case ofbare CNPs with the Aβ protein. The force histogram of Aβ with CNPs showsthat the force of the interaction is in the range of 10-250pN (204 forcecurve analyzed) (F). The interaction force of Aβ is found to be higherwith amine functionalized nanoparticles (2D) and force histogram shows(2G) that the amount of interaction is in the range of 250pN-500pN (320force carve analyzed), greater than the bare CNPs, which indicatesincreased non specific interaction to the protein. Interestingly,minimum or no interaction has been observed with PEG-CNPs (2E). Theinteraction force is in the order: NH2-CNP>Bare CNP>PEG-CNP. This isexplained in terms of zeta potential of the bare and functionalizednanoparticle. As the zeta potential is changed from positive to negative(NH2-CNP (+16 mV)<Bare CNPs (−10 mV)<PEG-CNP (−37 mV)), it minimizes theinteraction with the partially negatively charged C-terminal part of theβ-amyloid protein and the CNPs. Schematic diagram of CNP interactionwith AFM probe functionalized with Aβprotein is consistent with ourearlier studies dealing with tranferrin conjugated CNPs (24). It isnoteworthing that the SMFM data suggested that PEG attachment with CNPsdecreases the nonspecific interaction and its importance is discussedlater.

CNPs-Ab Targeting Plaques

In FIG. 3 while control cells are negative to the thioflavine T staining(B), the green fluorescence shows the presence of Aβ plaque inAβ-treated cells with (H) or without nanoparticles (E). The nuclei,stained with DAPI (D, G), show cell aggregation in the areas closed tothe Aβ plaque, while control cells appear dispersed (A). In the samefigure the ability of CNPs-Ab to bind the Aβ plaques is assessed bydouble immunostainining of thT and anti-rabbit Tritc-labeled secondaryantibody (C, F, I L merge) which specifically recognizes the antibodybound to the nanoparticles. Tritc-antibody labels only cells treatedwith both Aβ plaque and CNP-Ab (I, L merge) not the control (C) andAβ-treated (F) cells. Therefore, the ligation of the specific antibodyagainst Aβ to the nanoparticles allows the CNPs-Ab to specificallyrecognize the plaque, having minimum or no interaction with nearbyneuronal cells. This is supported by the effects observed when the sameantibody used in the ligation is utilized alone to treat the cells afterthe Aβ challenge (Suppl materials, Fig. S4); in fact, it identifies theplaque (A, B, C) but having minimum or no effect on cell viability (D).

Cell Viability and Death

Preliminary experiments (Suppl materials, Fig S5) the effects on cellviability of PEG-CNP or NH₂—CNP administered alone or after Aβ challengeindicated that PEG-CNP are more effective than NH₂—CNP in promotingneuronal survival due to the decrease in non-specific interaction in PEGCNP. CNPs-PEG-Ab were found to be more effective as compared to PEG-CNP.Therefore, in all subsequent experiments only the PEG-CNPs-Ab (CNPs-Ab)were used. Cell viability, evaluated by MTS assay, in control andtreated cells is shown in FIG. 4, panel A. Aβ treatment leads tosignificant reduction in cell viability. CNPs-Ab revert this effect tothe control values. In the same figure (panel B) apoptotic cell death,evaluated as nucleosome concentration, is shown. Aβ treatment induces asignificant increase in apoptotic cell death, while after CNPs-Abtreatment no significant differences are observed with respect to thecontrol. Panel C shows the nuclear fragmentation in control and treatedcells, evaluated by DAPI nuclear staining. Consistently with theapoptotic assay, Aβ treatment leads to an increase in apoptotic nuclei,while CNPs-Ab almost restores the control condition. These results,taken together, indicate that the CNPs-Ab play a protective effectagainst the Aβ cytotoxic insult.

Cell Morphology

FIG. 4, panel D, shows the contrast phase microscopy and the graphicrepresentation of neurite length and number in control and treatedcells. Control cells (CTR) show an evident neuronal clustering andneuronal aggregation, Aβ treatment induced an evident neurite loss (Aβ).CNPs-Ab protects cells from neurite atrophy (Aβ-CNP-Ab). The graphicalrepresentation of number of neurite (left side), and of neurite length(right side) shows that Aβ treatment significantly decreases neuritenumber and also the neurite length, while CNPs-Ab protect the neuritesfrom Aβ-mediated neuronal damage.

Immunofluorescence

Since we have previously demonstrated that Aβ treatment affected theexpression of peroxisome proliferator activated receptor β (PPARβ),which is a transcription factor in neuronal differentiation (25), and inneuronal maturation (26-27), in the following experiments we analyzedthe cytoskeletal organization and the PPARβ/δ expression andlocalization in control and treated cells. In FIG. 5, doubleimmunostaining for 13-tubulin III, a marker of early neuronaldifferentiation and PPARβ/δ is shown. In control cells, β-tubulin III(CTR) localizes at cytoplasmatic and neurite level, while PPARβ/δ ismainly localized to the nuclei (CTR, merge). After Aβ treatment theneurite network is no more evident (Aβ) and the PPARβ/δ is mainlylocalized to the cytoplasm (Aβ merge).

In the β-treated cells following CNPs-Ab an evident preservation ofneurite network (Aβ-CNP-Ab merge) as well as of the PPARβ/δ nuclearlocalization is observed (Aβ-CNP-Ab merge), thus indicating protectionof neurites by nanoceria. Regarding PPARβ/δ protein levels, Aβ treatmentsignificantly downregulates the protein, while CNPs-Ab revert PPARβ/δprotein levels to the control (FIG. 5 bottom).

In FIG. 6 the heavy neurofilament 200 (NF-H) and GAP-43 localization incontrol and treated cells is shown. NF-H is a marker of neuronalterminal maturation, while GAP-43 is an axonal marker. In control cellsa wide neuronal network is observed (A and D, G and L), while inAβ-treated cells the network is completely lost (B and E, H and M).After CNPs-Ab treatment an evident preservation of the neurites isobserved (C and F, I and N), thus confirming the neuroprotective effectsby the presently disclosed nanoceria composition in counteractingneuronal dystrophy.

Signal Transduction Pathways

Finally, since neuronal morphology and plasticity are correlated to thebrain derived neurotrophic factor (BDNF) signal transduction pathway, weassayed the BDNF, its receptors such as TrkB and p75, and theextracellular signal regulated kinases such as ERK1,2 and ERK5. Upon Aβchallenge, the cytoplasmatic levels of BDNF immature form (pro-BDNF)show to be upregulated (FIG. 7 panel B); The same results are obtainedby the immunoprecipitation assay of the culture media (FIG. 7 panel B),indicating that Aβ injury leads to a strong accumulation of pro-BDNF inthe extracellular matrix. This significant increase in pro-BDNF may beresponsible for the promotion of the neuronal death and atrophy, as itis known that the immature form of BDNF induces neuronal apoptosis viaactivation of a receptor complex of p75NTR and sortilin (28). This viewis supported by the results obtained for p75NTR protein in ourexperimental condition (FIG. 7 panel A). In fact, as for pro-BDNF, Aβincreases p75NTR protein levels while concomitantly triggering adecrease of the specific receptor TrkB involved in the action of matureand cleaved form of BDNF. Moreover Aβ induces the active form of ERK1,2(p-ERK1,2) (FIG. 7 panel C), known to be involved in apoptosispromotion. In Aβ-treated cells following CNPs-Ab a reduction but not acomplete reversion of pro-BDNF levels is observed, while the levels ofthe mature form are reverted to the control values. In the same time,CNPs-Ab, significantly increase TrkB as well as the p-ERK5, involved inneuronal survival, with concomitant decrease of ERK1,2, suggesting anactivation of the neuronal survival pathway BDNF/TrkB/ERK5.

Discussion

It has been previously reported that GNPs protect neurons from freeradical—mediated insult initiated by UV light, H₂O₂, irradiation, andexcitotoxicity (29-30). Our previous results have documented theanti-oxidant and protective role of bare cerium oxide nanoparticle in ahuman AD in vitro model.

In this work we have carefully designed and formulated a targetedpegylated nanoceria-based molecule suitable as therapy for AD. Design ofa specifically targeted nanoparticle avoids diffusion to other areas andin the cell cytoplasm. The conjugation of CNPs with an antibody againstAβ 1-42 makes possible a targeted delivery of CNPs to the Aβ plaques.

In this composite nanoparticle we have also used the 18.1 Å lengthbi-functional PEG as a spacer to conjugate anti Aβ antibody with aminefunctionalized CNPs. The SMFP data showed that the attachment of PEG toCNPs reduces non-specific interactions with Aβ proteins and protects theantioxidant properties of the CNPs while targeting the plaques. In thepresently disclosed composition PEG acts as a spacer which also providesflexibility to the conjugated antibody to interact with its ligand withminimal steric hinderance.

Moreover, PEG coating will also provide the following advantages apartfrom the specific targeted delivery: (i) causing high disparity ofnanoparticles; (ii) protecting nanoparticles from agglomerating andbeing cleared out from the system, (iii) minimizing the attachment ofopsonin protein and suppressing uptake by macrophages, and (iv)increasing blood circulation time (31).

The immunofluorescence results obtained indicate the specific targetingof nanoceria to Aβ plaques without diffusion to the cell cytoplasm. Itis noteworthy that the specific formulation now proposed is effective ata significantly lower concentration than bare CNPs which may decreasethe potential drug side-effects. Moreover CNPs-Ab other than exertingnon-specific antioxidant effects, seem to modulate at translationallevel proteins crucial for the neuronal signal transduction pathwayleading to survival, such as TrkB and p-ERK5, which appearedsignificantly upregulated, and the proapoptotic signaling proteins suchas BDNF/p75/p-ERK1,2, which appeared down-regulated. Consistently withthe increase of the survival and plasticity pathways, the neuronalcytoskeleton, strongly damaged by Aβ challenge, appeared completelypreserved in Aβ-treated cells in presence of CNPs-Ab.

Nanoparticles have been largely employed to deliver various types ofdrugs ranging from coenzyme Q10 (32), to protein antigens (33), plasmidDNA (34) and several other molecules. Specific nanoparticles weredemonstrated to penetrate the blood-brain barrier (BBB) without alteringits permeability and to be circulating in the blood for a long time(35). However, the inventive nanoceria composition disclosed hereindemonstrates the use of functionalized self targeting nanoceria bound toa specific carrier for counteracting brain pathologies characterized byoxidative stress, and has shown to be effective in counteracting diseaseprogression by improving neuronal viability while decreasing neuronaldeath and neurite atrophy.

Accordingly, in the drawings and specification there have been disclosedtypical preferred embodiments of the invention and although specificterms may have been employed, the terms are used in a descriptive senseonly and not for purposes of limitation. The invention has beendescribed in considerable detail with specific reference to theseillustrated embodiments. It will be apparent, however, that variousmodifications and changes can be made within the spirit and scope of theinvention as described in the foregoing specification and as defined inthe appended claims.

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That which is claimed:
 1. A composition comprising polyethylene glycol(PEG) coated nanoparticles of Cerium oxide having an antibody boundthereto, the antibody being specific for an antigen associated with apredetermined disease condition, wherein the PEG comprises abifunctional molecule comprising a carboxy terminal and amine terminal,the carboxy terminal being connected to an amine group on thenanoparticles and an amine terminal being connected to the antibody asfollows:CeO₂—NHCO—[CH₂CH₂O]^(n)—CH₂CH₂NH-antibody.
 2. The composition of claim1, wherein said nanoparticles are amine functionalized to promotecoating by the PEG.
 3. The composition of claim 1, wherein said antibodyis specifically targeted against an amyloid-beta antigen associated witha neurodegenerative disease.
 4. The composition of claim 1, wherein thenanoparticles are approximately from 3-5 nm in size prior to coatingwith PEG.
 5. The composition of claim 1, contained in a manufacturedmedication biologically acceptable for administration to a patientexhibiting symptoms of the predetermined disease.
 6. A compositionspecifically targeted to a neurodegenerative disease, said compositioncomprising amine functionalized nanoparticles of Cerium oxide coatedwith polyethylene glycol and bearing an antibody specific for an antigenassociated with the neurodegenerative disease; wherein the PEG comprisesa bifunctional molecule comprising a carboxy terminal and amineterminal, the carboxy terminal being connected to an amine group on thenanoparticles and an amine terminal being connected to the antibody asfollows:CeO₂—NHCO—[CH₂CH₂O]_(n)—CH₂CH₂NH-antibody.
 7. The composition of claim6, contained in a manufactured medication biologically acceptable foradministration to a patient exhibiting symptoms of the neurodegenerativedisease.
 8. A method of treatment for a neurodegenerative disease, themethod comprising administering the composition of claim 6 to a patientin need thereof.
 9. A composition immunologically targeted toAlzheimer's disease (AD), said composition comprising aminefunctionalized nanoparticles of Cerium oxide coated with polyethyleneglycol and bearing an antibody specific for an amyloid-beta antigenassociated with AD, wherein the PEG comprises a bifunctional moleculecomprising a carboxy terminal and amine terminal, the carboxy terminalbeing connected to an amine group on the nanoparticles and an amineterminal being connected to the antibody as follows:CeO₂—NHCO—[CH₂CH₂O]_(n)—CH₂CH₂NH-antibody.
 10. The composition of claim9, contained in a manufactured medication biologically acceptable foradministration to a patient suffering from AD.
 11. A method of treatmentfor Alzheimer's disease, the method comprising administering thecomposition of claim 9 to a patient suffering from AD.